Tumorigenesis arises when DNA damage exceeds DNA repair. The most common form of DNA damage is endogenous oxidative damage, which is repaired by base excision repair (BER), and in some cases nucleotide-excision repair (NER), homologous recombination (HR) and/or non- homologous end-joining (NHEJ). BRCA1, a 220kD tumor suppressor protein, has been implicated in preventing breast cancer by maintaining genetic stability through its ability to prevent oxidative DNA damage and promote DNA repair, including BER, NER, HR, and NHEJ. Upon mutation or inactivation of BRCA1, excessive DNA damage leads to the accumulation of mutations and genetic instability, and ultimately formation of BRCA1-associated malignancies. BRCA1-associated malignancies include hereditary breast and ovarian cancers due to mutations in BRCA1, sporadic breast cancers due to epigenetic silencing of BRCA1, and a subset of breast cancers with the basal-like phenotype (i.e. triple-negative for ER/PR/HER2), which have a similar expression profile to cancers with mutations in BRCA1. They comprise 30-50% of breast cancer cases. These malignancies often do not respond to current chemoprevention agents, suggesting a need for effective prevention strategies. This study will initiate a novel, targeted approach to preventing BRCA1-associated malignancies. The hypothesis states: high-throughput (HT) screening of a compound library may identify small molecules that activate DNA repair of oxidative DNA damage in the presence of aberrant BRCA1. Lead compounds can then be used to identify potential chemoprevention agents for BRCA1-associated malignancies or as tools in basic science research. The following specific aims will be addressed: (1) validate a DNA repair assay for high- throughput format., (2) carry out a quantitative high-throughput pilot screen using a DNA repair assay, and (3) carry out a quantitative high-throughput counter screen using an oxidative DNA damage response assay. First, a cell-based assay that uses adenovirus containing oxidative DNA damage within the coding region of a GFP reporter gene to monitor for activation of DNA repair by host-cell reactivation will be developed for HT-screening. Therefore, optimal cell growth and treatment conditions will be determined, and plate acceptance for the DNA repair assay will be carried out in 384-well format. Second, a small, diverse compound library and the quantitative high-throughput screening format will be used to carry out a pilot screen and to determine assay reproducibility. Finally, we will adapt and validate an H2O2 sensitivity assay for high-throughput format, test the assay in a quantitative high-throughput pilot screen, and compare the hits obtained from the pilot screens using the DNA repair and H2O2 sensitivity assays. Overall, a DNA repair assay will be developed, adapted, and validated for high-throughput screening of a compound library to identify potential chemoprevention agents for BRCA1-associated malignancies. PUBLIC HEALTH RELEVANCE: We will initiate the discovery of potential drugs for preventing a subset of breast cancer known as BRCA1-associated malignancies. Furthermore, this study may lead to advances in treating other diseases that result from oxidative DNA damage (e.g. repair-deficient cancers, degenerative diseases, and various sclerotic diseases), and may generate tools for basic science research.